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Biophys J. 2016 May 24;110(10):2147-50. doi: 10.1016/j.bpj.2016.04.011. Epub 2016 May 10.

Assembly and Comparison of Plasma Membrane SNARE Acceptor Complexes.

Author information

1
Center for Membrane and Cell Physiology and Department of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, Virginia.
2
Center for Membrane and Cell Physiology and Department of Molecular Physiology & Biological Physics, University of Virginia, Charlottesville, Virginia. Electronic address: lkt2e@virginia.edu.

Abstract

Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes. Here we present two (to our knowledge) new procedures to assemble membrane-bound 1:1 SNARE acceptor complexes that produce fast and efficient fusion without the need of the syb(49-96) peptide. In the first procedure, syntaxin-1a is purified in a strictly monomeric form and subsequently assembled with SNAP-25 in detergent with the correct 1:1 stoichiometry. In the second procedure, monomeric syntaxin-1a and dodecylated (d-)SNAP-25 are separately reconstituted into proteoliposomes and subsequently assembled in the plane of merged target lipid bilayers. Examining single particle fusion between synaptobrevin-2 proteoliposomes and planar-supported bilayers containing the two different SNARE acceptor complexes revealed similar fast rates of fusion. Changing the stoichiometry of syntaxin-1a and d-SNAP-25 in the target bilayer had significant effects on docking, but little effect on the rates of synaptobrevin-2 proteoliposome fusion.

PMID:
27178662
PMCID:
PMC4881159
DOI:
10.1016/j.bpj.2016.04.011
[Indexed for MEDLINE]
Free PMC Article

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